Since the description by deBold et al, Life Sci 28 89-94 (1981) of a potent atrial natriuretic factor extracted from rat atria, considerable attention has been directed to a family of diuretic and natriuretic peptides which have been shown to be stored in and secreted from mammalian atria. Attention is directed to deBold U.S. Pat. No. 4,663,437 issued May 5, 1987, Thibault et al U.S. Pat. Nos. 4,607,023 issued Aug. 19, 1986 and 4,455,864 issued Jan. 29, 1985 and Dec. 10, 1985 respectively. These references all teach atrial peptides having up to 71 amino acid residues in a sequence which always includes: Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Se r-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr or minor variations thereof depending on the atrial source (rat, human, etc.) and the precise method of isolation thereof. DeBold also established the existence of a disulphide bond between the cysteine residues and consequently the looped structure of the peptide. Recent sequence determinations by Needleman at al have shown (Science 1985; 229:397-400) the 28 amino acid peptide to be the circulating form of the peptide in humans. The human 28 amino acid peptide differs from the rat sequence only at position 110 at which position methionine occurs in the human peptide and isoleucine occurs in the rat peptide. These peptides, variously named ANF, cardionatrin, auriculin or atriopeptin, depending upon the authority, have been shown to exhibit profound diuretic, natriuretic and hypotensive effects of relatively short duration when injected into non-diuretic mammalian subjects, including man. These peptides are, therefore, of considerable interest and utility for therapeutic applications despite the lack of oral bioavailability and short half life of currently known analogs.
Initially ANF was produced by carboxylic extraction of rat heart atria, but as the amino-acid sequences were developed, most ANF peptides may now be produced synthetically. In the carboxylic acid extraction rat atria, either freshly dissected or frozen were obtained from standard commercial sources (i.e. diet of the rat is unimportant), finely ground in liquid nitrogen and extracted with a mixture of acetic acid and HCl containing phenyl methyl sufonyl fluoride in a homogenizer. After centrifugation the supernatant was passed through Sep-Pak cartridges and the cartridges were washed with trifluoroacetic acid (TFA) and eluted with acetonitrile in TFA. the eluates were reduced in volume and again separated on a C.sub.18 column. The column was eluted with acetonitrite at different gradients.
Radioimmunoassays for ANF have also been developed in conventional manner by injection of chemically synthesized peptide, coupled to thyroglobulin, into rabbits for production of specific antisera. The antisera has then been combined into specific RIAs.
In the present specification the symbols for amino acids are according to standard IUPAC-IUB recommendations and single-letter symbols and three-letter symbols are used interchangeably for convenience according to the following table:
______________________________________ A Ala Alanine B Asx Asparagine or aspartic acid C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine Z Glx Glutamine or glutamic acid ______________________________________